WO2017169399A1 - Transformant, et procédé de production d'acide protocatéchuique ou son sel l'utilisant - Google Patents

Transformant, et procédé de production d'acide protocatéchuique ou son sel l'utilisant Download PDF

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WO2017169399A1
WO2017169399A1 PCT/JP2017/007233 JP2017007233W WO2017169399A1 WO 2017169399 A1 WO2017169399 A1 WO 2017169399A1 JP 2017007233 W JP2017007233 W JP 2017007233W WO 2017169399 A1 WO2017169399 A1 WO 2017169399A1
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乾将行
平賀和三
須田雅子
小暮高久
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公益財団法人地球環境産業技術研究機構
住友ベークライト株式会社
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Priority to JP2018508800A priority Critical patent/JP6685388B2/ja
Priority to US16/089,567 priority patent/US10961526B2/en
Priority to CN201780021444.5A priority patent/CN109477066B/zh
Priority to EP17773944.8A priority patent/EP3438245B1/fr
Publication of WO2017169399A1 publication Critical patent/WO2017169399A1/fr

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13064—4-Hydroxybenzoate 1-hydroxylase (1.14.13.64)
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    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/03—Oxo-acid-lyases (4.1.3)
    • C12Y401/0304—Chorismate lyase (4.1.3.40)
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01118—3-Dehydroshikimate dehydratase (4.2.1.118)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the present invention relates to a transformant capable of efficiently producing protocatechuic acid or a salt thereof using a saccharide as a raw material by performing a specific genetic manipulation, and an efficient protocatechu using the transformant.
  • the present invention relates to a method for producing an acid.
  • Protocatechuic acid is a useful compound used as an antioxidant in addition to being a raw material for pharmaceuticals, agricultural chemicals, fragrances and the like.
  • protocatechuic acid is mainly produced by extraction from natural products (agricultural products).
  • natural products agricultural products
  • Patent Documents 1 and 2 describe that a Escherichia bacterium or Klebsiella bacterium that can convert a carbon source into 3-dehydroshikimate via a common aromatic amino acid biosynthesis pathway, a 3-dehydroshikimate dehydratase gene derived from a Klebsiella bacterium, And a method for producing catechol from saccharides via protocatechuic acid using a transformant introduced with a protocatechuate decarboxylase gene.
  • Patent Document 2 further teaches that for the production of catechol via protocatechuic acid, it is preferable to inhibit the conversion of 3-dehydroshikimate to chorismate by inactivating shikimate dehydrogenase. ing.
  • Patent Documents 3 and 4 use a transformant in which a 3-dehydroshikimate dehydratase gene, a protocatechuate decarboxylase gene, and a catechol 1,2-dioxygenase gene are introduced into a bacterium of the genus Escherichia or Klebsiella. Teaches how to produce cis, cis-muconic acid or adipic acid from saccharides via protocatechuic acid. Patent Documents 4 and 5 teach that it is preferable to inhibit any enzyme on the metabolic pathway from 3-dehydroshikimic acid to chorismic acid.
  • Patent Document 5 discloses protocatechuic acid from saccharides using a transformant in which a 3-dehydroshikimate dehydratase gene and a mutant 4-hydroxybenzoate hydroxylase gene are introduced into a bacterium belonging to the genus Escherichia or Klebsiella. Teaches how to make gallic acid or pyrogallol.
  • Patent Documents 1 to 5 do not intend to produce protocatechuic acid, and the problem is that the produced protocatechuic acid is converted into catechol, cis, cis-muconic acid, adipic acid, or gallic acid. is there. In addition, these substances cannot be produced sufficiently efficiently in practice.
  • An object of the present invention is to provide a microorganism capable of efficiently producing protocatechuic acid or a salt thereof from a saccharide, and a method for efficiently producing protocatechuic acid or a salt thereof using the microorganism.
  • Protocatechuic acid is generally known to be cytotoxic to microorganisms, and the possibility that productivity was limited by the toxicity of the produced protocatechuic acid was considered. Therefore, we compared the effects of protocatechuic acid on the growth of some microorganisms that have been reported to produce aromatic compounds so far. Among erythropolis, Corynebacterium glutamicum was shown to be the most resistant to protocatechuic acid.
  • Corynebacterium glutamicum showed high growth ability and sugar consumption ability even in the presence of a high concentration of protocatechuic acid of 500 mM, in which the growth of other microorganisms was completely or significantly suppressed.
  • Corynebacterium glutamicum is particularly suitable for the production of protocatechuic acid or a salt thereof because of its extremely high resistance to protocatechuic acid.
  • the coryneform bacterial transformant subjected to both (a) and (b) significantly improves the productivity of protocatechuic acid or its salt while blocking the aromatic amino acid biosynthetic pathway. Therefore, there is no requirement for aromatic amino acids such as ⁇ lipptophan, tyrosine, and phenylalanine, and paraaminobenzoic acid, so that it is not necessary to add these compounds to the medium to grow the transformant.
  • This transformant has a particularly high production efficiency of protocatechuic acid or a salt thereof when the reaction is carried out under an aerobic and substantially non-proliferating condition.
  • the present invention has been completed based on the above findings, and provides the following transformant and a method for producing protocatechuic acid or a salt thereof.
  • Item 1 A transformant capable of producing protocatechuic acid, which has been subjected to the following operations (A), (B), and (C).
  • a transformant capable of producing protocatechuic acid which has been subjected to the following operations (A), (B), and (C).
  • B Enhancement of chorismate pyruvate lyase activity
  • C Enhancement of 4-hydroxybenzoate hydroxylase activity 2.
  • the genes encoding enzymes with 3-dehydroshikimate dehydratase activity are Corynebacterium glutamicum, Corynebacterium halotolerans, Corynebacterium casei, Corynebacterium efficiens (Corynebacterium Item 3.
  • the transformant according to Item 2 or 3, wherein the gene encoding an enzyme having 3-dehydroshikimate dehydratase activity is encoded by the following DNA (a) or (b): (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 7, 134, 135, 145, 147, or 149 (b) a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 7, 134, 135, 145, 147, or 149, comprising a polypeptide having 3-dehydroshikimate dehydratase activity DNA to encode Item 5.
  • the enhancement of the chorismate pyruvate lyase activity is caused by the introduction of a gene encoding an enzyme having chorismate pyruvate lyase activity derived from Providencia or Cronobacter bacterium into the host.
  • the transformant according to any one of 1 to 4.
  • Item 6 Host of genes whose enhanced chorismate pyruvate lyase activity encodes an enzyme with chorismate pyruvate lyase activity from Providencia rustigianii, Providencia stuartii, or Cronobacter sakazakii Item 6.
  • the transformant according to Item 5 which is caused by introduction into the cell.
  • Item 7. Item 7.
  • the enhancement of 4-hydroxybenzoate hydroxylase activity was caused by the introduction of the gene for Corynebacterium glutamicum, which encodes an enzyme having 4-hydroxybenzoate hydroxylase activity, into the host.
  • the transformant according to any one of Items 1 to 7. Item 9.
  • the enhancement of the DAHP synthase activity was brought about by the introduction of the DNA of the following (g) or (h) into the host, and the enhancement of the 3-dehydroquinate synthase activity was the following (i) or (j) And the enhancement of 3-dehydroquinate dehydratase activity was caused by the introduction of DNA of (k) or (l) below into the host.
  • the enhancement of acid dehydrogenase activity was brought about by introduction of the following DNA of (m) or (n) into the host, and the enhancement of shikimate kinase activity was achieved by the following DNA of (o) or (p):
  • the enhancement of EPSP synthase activity was caused by introduction of the DNA of the following (q) or (r) into the host, and enhancement of chorismate synthase activity
  • the following (s) or (t) DNA may be introduced into the host Item 12.
  • Item 13 The transformant according to any one of Items 1 to 12, wherein at least one activity selected from the group consisting of transketolase activity and transaldolase activity is enhanced.
  • Item 14 The enhancement of transketolase activity is due to the introduction of the following DNA (u) or (v), and the enhancement of transaldolase activity is due to the introduction of the following DNA (w) or (x): The transformant according to 13.
  • Item 17. The transformant according to Item 15 or 16, wherein the host coryneform bacterium is a genus Corynebacterium. Item 18. Item 18. The transformant according to Item 17, wherein the host Corynebacterium bacterium is Corynebacterium glutamicum. Item 19. Item 19. The coryneform bacterium transformant according to Item 18, wherein the host Corynebacterium glutamicum is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869. Item 20. Corynebacterium glutamicum PCA4 (Accession number: NITE BP-02217) Item 21. Item 21.
  • a method for producing protocatechuic acid or a salt thereof comprising a step of culturing the transformant according to any one of Items 1 to 20 in a reaction solution containing a saccharide to produce protocatechuic acid or a salt thereof.
  • Item 22. The method according to Item 21, wherein the transformant is cultured under aerobic conditions where the transformant does not grow.
  • the biosynthesis pathway of protocatechuic acid in microorganisms includes (a) a protocatechuic acid production pathway by conversion of 3-dehydroshikimic acid to protocatechuic acid, catalyzed by 3-dehydroshikimate dehydratase, b) Two pathways, chorismate pyruvate lyase and protocatechuate production pathway by conversion of chorismate (the final metabolite of shikimate pathway) to protocatechuate catalyzed by 4-hydroxybenzoate hydroxylase Exists.
  • the two metabolic pathways (a) and (b), which branch from 3-dehydroshikimic acid as a base point and lead to the production of protocatechuic acid, are strengthened at the same time.
  • Protocatechuic acid production is significantly increased. That is, in coryneform bacteria, (a) 3-dehydroshikimate dehydratase activity, (b) chorismate pyruvate lyase activity, and 4-hydroxybenzoate hydroxylase activity are simultaneously applied (a ) Or only (b), the production amount of protocatechuic acid or a salt thereof from the saccharide is synergistically improved.
  • Coryneform bacteria have genes on the chromosome that encode 3-dehydroshikimate dehydratase and 4-hydroxybenzoate hydroxylase among the above three enzymes, but encode chorismate pyruvate lyase. It has no gene to do.
  • protocatechuic acid useful as a raw material for pharmaceuticals, fragrances, polymers and the like can be produced at low cost and in large quantities by a fermentation method with a low environmental load.
  • the growth of microorganisms is inhibited by the cytotoxicity of aromatic compounds such as protocatechuic acid, it has been difficult to efficiently produce protocatechuic acid using microorganisms.
  • coryneform bacteria have a very high resistance to aromatic compounds containing protocatechuic acid, a high concentration of protocatechuic acid or a salt thereof can be efficiently produced using the transformant of the present invention.
  • Coryneform bacteria unlike E.
  • FIG. 1 schematically shows a protocatechuic acid biosynthesis pathway in a coryneform bacterium transformant.
  • a transformant capable of producing protocatechuic acid or a salt thereof Host
  • any microorganism having an ability to produce protocatechuic acid can be used as a host.
  • Suitable host microorganisms include Corynebacterium bacteria, Escherichia bacteria (especially Escherichia coli), Bacillus bacteria (especially Bacillus subtilis), Pseudomonas bacteria (especially Pseudomonas putida), Brevibacterium bacteria, Streptococcus bacteria , Lactobacillus bacteria, Rhodococcus bacteria (especially Rhodococcus erythropolis, Rhodococcus opacus), Streptomyces bacteria, Saccharomyces yeasts (especially Saccharomyces cerevisiae), Klaveromyces yeasts, Schizosaccharomyces yeasts, Yarrowia Examples include yeast, Trichosporon yeast, Rhodosporidium yeast, Pichia yeast, Candida yeast, Neurospora mold, Aspergillus mold, Trichoderma mold and the like.
  • coryneform bacteria are preferably used as the host in terms of production efficiency of protocatechuic acid or a salt thereof.
  • Coryneform bacteria are a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), under normal aerobic conditions. If it proliferates, it will not be specifically limited. Specific examples include Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Micrococcus, and the like. Among the coryneform bacteria, the genus Corynebacterium is preferable.
  • Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanoler Examples include riticam (Corynebacterium alkanolyticum). Of these, Corynebacterium glutamicum is preferable because it is safe and has high protocatechuic acid productivity.
  • Corynebacterium glutamicum R strain (FERM BP-18976), ATCC13032 strain, ATCC13869 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 strain, ATCC13287 strain, ATCC13655 strain, ATCC13745 Strains, ATCC13746 strain, ATCC13761 strain, ATCC14020 strain, ATCC31831 strain, MJ-233 (FERM BP-1497), MJ-233AB-41 (FERM BP-1498) and the like.
  • Corynebacterium glutamicum strains are deposited internationally under the Budapest Treaty and are publicly available. Among these, R strain (FERM BP-18976), ATCC13032 strain, and ATCC13869 strain are preferable.
  • corynetypes such as Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, Corynebacterium lilium, etc.
  • the name of the bacterium is the same as Corynebacterium glutamicum (Liebl, W. et al., Trans Brevibacterium divaricatum DSM 20297T, "Brevibacterium flavum” DSM 20411, "Brevibacterium lactofermentum DSM DSM DSM DSM DSM , And Corynebacterium glutamicum and their distinction by rRNA gene restriction patterns.
  • Corynebacterium glutamicum Int J Syst Bacteriol. 41: 255-260. (1991), Komagata Kazuo et al., Coryneform bacteria classification, fermentation and industry, 45: 944-963 (1987) ].
  • Examples of the genus Brevibacterium include Brevibacterium ammoniagenes (for example, ATCC6872 strain).
  • examples of the genus Arthrobacter include Arthrobacter globiformis (for example, ATCC 8010 strain, ATCC 4336 strain, ATCC 21056 strain, ATCC 31250 strain, ATCC 31738 strain, ATCC 35698 strain) and the like.
  • Examples of the genus Mycobacterium include Mycobacterium bovis (for example, ATCC19210 strain, ATCC27289 strain).
  • Examples of the genus Micrococcus include Micrococcus freudenreichii (for example, No. 239 strain (FERM P-13221)), Micrococcus leuteus (for example, No.
  • Examples include Micrococcus ureae (for example, IAM1010 strain), Micrococcus roseus (for example, IFO3764 strain), and the like. These Brevibacterium, Arthrobacter, Mycobacterium, and Micrococcus strains are internationally deposited under the Budapest Treaty and are publicly available.
  • the coryneform bacterium may be a mutant strain or an artificial gene recombinant.
  • disrupted strains of genes such as lactate (lactate dehydrogenase: LDH), phosphoenolpyruvate carboxylase, and malate dehydrogenase.
  • lactate dehydrogenase lactate dehydrogenase: LDH
  • phosphoenolpyruvate carboxylase phosphoenolpyruvate carboxylase
  • malate dehydrogenase a disrupted strain of lactate dehydrogenase gene is preferable. In this gene-disrupted strain, the metabolic pathway from pyruvate to lactic acid is blocked because the lactate dehydrogenase gene is disrupted.
  • a disrupted strain of Corynebacterium glutamicum particularly a lactate dehydrogenase gene of R (FERM BP-18976) strain is preferable.
  • a gene-disrupted strain can be prepared according to a conventional method by genetic engineering techniques.
  • WO2005 / 010182A1 describes a lactate dehydrogenase-disrupting strain and a method for producing the same.
  • coryneform bacteria are extremely resistant to protocatechuic acid compared to other bacteria. Further, as shown in FIG. 3, the coryneform bacterium exhibited a high sugar consumption ability even in the presence of a high concentration of protocatechuic acid. In these respects, coryneform bacteria are suitable for producing protocatechuic acid or a salt thereof by the method of the present invention.
  • Transformants produce better protocatechuic acid transgene present invention efficiently in a host strain, 3-dehydroshikimic acid dehydratase, to strengthen the Collis formate pyruvate lyase, and 4-hydroxybenzoic acid hydroxy each enzyme activity of hydrolase Can be obtained.
  • 3-dehydroshikiate dehydratase catalyzes the reaction of producing protocatechuic acid from 3-dehydroshikimate.
  • Chorismate pyruvate lyase catalyzes the reaction that produces 4-hydroxybenzoic acid from chorismate.
  • 4-hydroxybenzoic acid hydroxylase catalyzes a reaction for producing protocatechuic acid by hydroxylating the 3-position carbon atom of the aromatic ring of 4-hydroxybenzoic acid.
  • the activity of these enzymes can be enhanced by introducing a gene encoding these enzymes into a host microorganism.
  • the activity enhancement of these enzymes can also be brought about by introducing a mutation into the control sequence of the enzyme gene present on the chromosome of the host microorganism, the gene coding region, or both, or by replacing the base sequence. Of these, it is simple and efficient to enhance the enzyme activity by introducing these enzyme genes into the host microorganism.
  • the bacterium When a coryneform bacterium is used as a host, the bacterium has a 3-dehydroshikimate dehydratase gene and a 4-hydroxybenzoate hydroxylase gene on the chromosome, but does not have a chorismate pyruvate lyase gene. Absent.
  • the 3-dehydroshikimate dehydratase gene and the 4-hydroxybenzoate hydroxylase gene are also expressed only under specific culture conditions (in the presence of protocatechuic acid or a specific aromatic compound). It is possible that Therefore, the above three genes are preferably introduced into the host coryneform bacterium as a fusion gene placed under the control of an appropriate promoter that provides high expression under the culture conditions used.
  • each gene is not particularly limited, for example, the following microorganism genes are mentioned in terms of good production efficiency of protocatechuic acid or a salt thereof.
  • the 3-dehydroshikimate dehydratase gene includes bacteria belonging to the genus Corynebacterium (in particular, Corynebacterium glutamicum, Corynebacterium casei), Corynebacterium efficience (Corynebacterium efficience) ), Corynebacterium halotolerans), Rhodococcus bacteria (especially Rhodococcus opacus), Mycobacterium bacteria (especially Mycobacterium smegmatis), Bacillus bacteria ( In particular, Bacillus thuringiensis), Gluconobacter bacterium (especially Gluconobacter oxydans), Rhodopseudomonas bacterium (especially Rhodopseudomonas spp.).
  • Corynebacterium in particular, Corynebacterium glutamicum, Corynebacterium casei), Corynebacterium efficience (Corynebacterium efficience) ), Corynebacterium halotolerans),
  • genes of Corynebacterium glutamicum, Corynebacterium casei, Corynebacterium efficiens, Corynebacterium halotolerance, Rhodococcus opacus, Methylobacterium exotolquiens, Neurospora crassa, Aspergillus niger, and Aspergillus oryzae are preferable. More preferred are genes of Corynebacterium glutamicum and Corynebacterium halotolerance.
  • the 3-dehydroshikimate dehydratase gene of Corynebacterium glutamicum of SEQ ID NO: 7 is called qsuB. Further, it is a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to any one of SEQ ID NO: 7 and SEQ ID NOs: 134 to 150, and has 3-dehydroshikimate dehydratase activity. DNA encoding a polypeptide having the same can also be used.
  • stringent conditions refers to hybridization in a 6 ⁇ SSC salt concentration hybridization solution at a temperature of 50 to 60 ° C. for 16 hours in a 0.1 ⁇ SSC salt concentration solution. This is the condition for cleaning.
  • DNA comprising a nucleotide sequence having 90% or more, particularly 95% or more, especially 98% or more identity with any one of SEQ ID NO: 7 and SEQ ID NOs: 134 to 150, and 3-dehydro
  • a DNA encoding a polypeptide having shikimate dehydratase activity can also be used.
  • the identity of the base sequence is a value calculated by GENETYX® ver. 8 (manufactured by GENETYX® GENETICS).
  • the enhancement of 3-dehydroshikimate dehydratase activity of the transformant is confirmed by measuring the 3-dehydroshikimate dehydratase activity in the cell extract of the transformant.
  • Collis formate pyruvate lyase gene Collis formate pyruvate but not limited lyase gene derived, in particular, in terms of good production efficiency of protocatechuic acid or a salt thereof, Providencia spp or a gene Chrono Enterobacter bacteria are preferred, among them, The gene of Providencia rustigianii, Providencia stuartii, and Cronobacter sakazakii are more preferred, and the gene of Providencia rustigianii is even more preferred.
  • the chorismate pyruvate lyase genes of Providencia rustigianii, Providencia stuartii, and Cronobacter sakazakii consist of the nucleotide sequences shown in SEQ ID NOs: 9, 128, and 129, respectively. Is mentioned.
  • the chorismate pyruvate lyase gene of Providencia rustigianii of SEQ ID NO: 9 is called ubiC.
  • the encoding DNA can also be used.
  • DNA comprising a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more of the nucleotide sequence of any one of SEQ ID NOs: 9, 128 and 129, and has a chorismate pyruvate lyase activity.
  • a DNA encoding a polypeptide having can also be used.
  • the chorismate pyruvate lyase activity is measured using a method modified from the method described in “Journal of Bacteriology, 174, 5309-5316, 1992“ Materials and Methods ””. That is, by adding a test enzyme solution to a reaction mixture consisting of 50 mM Tris-HCl buffer (pH 7.5), 20 mM NaCl, 0.2 mM NADH, 0.5 mM chorismate, 5 U / ml lactate dehydrogenase at 33 ° C. Beckman DU 800 spectrophotometer The enzyme activity is calculated from the initial reaction rate. The activity at which 1 ⁇ mol of NADH is consumed per minute at 33 ° C.
  • the enhancement of the chorismate pyruvate lyase activity of the transformant is confirmed by an increase in the chorismate pyruvate lyase activity in the cell extract of the transformant.
  • 4-Hydroxybenzoate hydroxylase gene 4-Hydroxybenzoate hydroxylase is also referred to as phenol monooxygenase.
  • the origin of the 4-hydroxybenzoate hydroxylase gene is not particularly limited, but the gene of Corynebacterium genus bacteria, especially the gene of Corynebacterium glutamicum (Corynebacterium glutamicum) is particularly advantageous in terms of good production efficiency of protocatechuic acid or its salts. preferable.
  • Examples of the 4-hydroxybenzoate hydroxylase gene of Corynebacterium glutamicum include those consisting of the base sequence shown in SEQ ID NO: 8.
  • the Corynebacterium glutamicum 4-hydroxybenzoate hydroxylase gene is called pobA.
  • DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of SEQ ID NO: 8 and that encodes a polypeptide having 4-hydroxybenzoate hydroxylase activity can also be used. .
  • polypeptide having a 4-hydroxybenzoic acid hydroxylase activity which is a DNA comprising a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more of the nucleotide sequence of SEQ ID NO: 8 DNA can also be used.
  • DAHP 3-deoxy-D-arabino-heptulosonate-7-phosphate
  • the transformant of the present invention further comprises 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase activity. Is preferably enhanced.
  • DAHP synthase is an enzyme that generates DAHP, which is an initial metabolite of an aromatic compound biosynthesis pathway, from erythrose-4-phosphate and phosphoenolpyruvate.
  • Enhancement of DAHP synthase activity can be achieved by introducing the DAHP synthase gene into the host microorganism, or introducing a mutation into the DAHP synthase gene (regulatory sequence and / or region, gene coding region, or both) on the chromosome of the host microorganism. Can bring. Of these, it is simple and efficient to enhance DAHP synthase activity by introducing a DAHP synthase gene into a host microorganism.
  • the origin of the DAHP synthase gene to be introduced is not particularly limited, but a gene derived from Corynebacterium glutamicum or Escherichia coli is preferable from the viewpoint of good production efficiency of protocatechuic acid or a salt thereof. Of these, genes derived from Escherichia coli are more preferable.
  • DNA (aroG S180F ) comprising the nucleotide sequence of SEQ ID NO: 2 is even more preferable.
  • This gene is a gene in which the mutation (S180F) that mutates the 180th serine of the amino acid sequence encoded by this gene to phenylalanine is introduced into the aroG gene, which is one of the DAHP synthase genes derived from Escherichia coli.
  • the present inventors have found through comparative studies that gene products exhibit resistance to feedback inhibition by aromatic compounds containing aromatic amino acids and high DAHP synthase activity (unpublished).
  • a DNA consisting of a nucleotide sequence having 90% or more, particularly 95% or more, especially 98% or more of identity with SEQ ID NO: 2, and a DNA encoding a polypeptide having DAHP synthase activity, or A DNA that hybridizes with a DNA comprising a base sequence complementary to SEQ ID NO: 2 under stringent conditions and that encodes a polypeptide having DAHP synthase activity can also be used.
  • DAHP synthase activity as follows. Reaction by adding the test enzyme solution to the reaction mixture consisting of 20 mM bistrispropane buffer (pH 6.8), 500 ⁇ M ⁇ phosphoenolpyruvate (PEP) sodium, 500 ⁇ M erythrose-4-phosphate, 1 mM manganese chloride
  • the activity at which 1 ⁇ mol of PEP is consumed per minute at 33 ° C. is defined as 1 ⁇ unit of DAHP synthase activity.
  • the enhancement of DAHP synthase activity of the transformant is confirmed by an increase in the DAHP synthase activity value in the cell extract of the transformant.
  • the transformant of the present invention preferably further has enhanced transketolase activity, or transketolase activity and transaldolase activity.
  • transketolase catalyzes two reactions.
  • the first reaction is the conversion of D-xylulose-5-phosphate to glyceraldehyde-3-phosphate and D-ribose-5-phosphate (R5P) in the non-oxidative pentose-phosphate pathway. It is a reaction that catalyzes the conversion of sucrose to sedheptulose-7-phosphate (S7P). These reactions are reversible and conjugated.
  • the second reaction consists of the conversion of D-fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P), and glyceraldehyde-3-phosphate to D-xylulose-5. A reaction that catalyzes the conversion to phosphoric acid. These reactions are reversible and conjugated.
  • transaldolase In sugar metabolism, transaldolase is converted from glyceraldehyde-3-phosphate to erythrose-4-phosphate, and from sedoheptulose-7-phosphate to D-fructose-6-phosphate. To catalyze. These reactions are conjugated.
  • transketolase and transaldolase play an important role in the production of erythrose-4-phosphate, which is one of the precursors of aromatic compound biosynthesis. Therefore, by enhancing these enzyme activities, the supply of intracellular erythrose-4-phosphate is increased, resulting in an increased metabolic flux into the aromatic compound biosynthetic pathway and an improvement in protocatechuic acid productivity. It is thought to bring.
  • the transketolase activity and the enhancement of the transaldolase activity can be achieved by introducing the transketolase gene and the transaldolase gene into the host microorganism or the control sequence of the transketolase gene or transaldolase gene on the chromosome of the host microorganism. , By introducing mutations into the gene coding region or both, and by sequence substitution. Of these, it is simple and efficient to enhance the enzyme activity by introducing the transketolase gene and transaldolase gene into the host microorganism.
  • the transketolase gene to be introduced and the origin of the transaldolase gene are not particularly limited, but in terms of good production efficiency of protocatechuic acid or a salt thereof, the transketolase gene of Corynebacterium, particularly Corynebacterium glutamicum, and A transaldolase gene is preferred.
  • Examples of the transketolase gene of Corynebacterium glutamicum include DNA (tkt) having the base sequence of SEQ ID NO: 151
  • examples of the transaldolase gene of Corynebacterium glutamicum include DNA (tal) having the base sequence of SEQ ID NO: 152 ).
  • a DNA comprising a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more of identity with SEQ ID NO: 151 or 152, and transketolase activity or transaldolase activity, respectively. It is also possible to use DNA encoding a polypeptide having Further, in the present invention, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to SEQ ID NO: 151 or 152, and encodes a polypeptide having transketolase activity or transaldolase activity, respectively. Can also be used.
  • the activity that consumes 1 ⁇ mol of NADH per minute at 33 ° C. is defined as 1 unit of transketolase activity, and it is determined that there is transketolase activity when the activity is detected.
  • the enhancement of the transketolase activity of the transformant is confirmed by an increase in the transketolase activity value in the cell extract of the transformant.
  • the activity at which 1 ⁇ mol of NADH is consumed per minute at 33 ° C. is defined as 1 unit of transaldolase activity. If the activity is detected, it is determined that there is transaldolase activity. In the present invention, the enhancement of the transaldolase activity of the transformant is confirmed by an increase in the transaldolase activity value in the cell extract of the transformant.
  • Enhancing the enzyme activities of 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, and chorismate synthase The transformant further comprises a series of enzymes on the shikimate pathway after DAHP synthase: 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, 5-enolpyruvylshikimate 3 -Preferably, at least one of the enzyme activities of phosphate (EPSP) synthase and chorismate synthase is enhanced, and more preferably, all of these enzyme activities are enhanced.
  • EBP phosphate
  • 3-dehydroquinic acid synthase is an enzyme that catalyzes the conversion of DAHP to 3-dehydroquinic acid
  • 3-dehydroquinic acid dehydratase is an enzyme that catalyzes the conversion of 3-dehydroquinic acid to 3-dehydroshikimic acid.
  • Acid dehydrogenase is an enzyme that catalyzes the conversion of 3-dehydroshikimate to shikimate
  • shikimate kinase is an enzyme that catalyzes the conversion of shikimate to shikimate-3-phosphate
  • EPSP synthase An enzyme that catalyzes the conversion of acid-3-phosphate to EPSP
  • chorismate synthase is an enzyme that catalyzes the conversion of EPSP to chorismate.
  • Enhancement of each enzyme activity of 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, EPSP synthase, and chorismate synthase can be achieved by introducing a gene encoding each of the enzymes into a host microorganism, or The gene can be brought about by introducing a mutation into the regulatory sequence of the enzyme gene on the chromosome of the host microorganism, the gene coding region, or both, and by replacing the nucleotide sequence. Of these, it is simple and efficient to enhance the enzyme activity encoded by introduction of each enzyme gene into a host microorganism.
  • each gene encoding 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, EPSP synthase, and chorismate synthase to be introduced is not particularly limited, but the production efficiency of protocatechuic acid and its salts However, it is preferably a gene of Corynebacterium, particularly Corynebacterium glutamicum.
  • the 3-dehydroquinate synthase gene includes DNA (aroB) consisting of SEQ ID NO: 153, and the 3-dehydroquinate dehydratase gene is DNA consisting of SEQ ID NO: 5 (aroD
  • the shikimate dehydrogenase gene includes DNA (aroE) consisting of SEQ ID NO: 6, the shikimate kinase gene includes DNA (aroK) consisting of SEQ ID NO: 154, and the EPSP synthase gene includes SEQ ID NO: And a chorismate synthase gene includes DNA (aroC) consisting of SEQ ID NO: 156.
  • DNA comprising a nucleotide sequence having the identity of SEQ ID NO: 153, 5, 6, 154, 155, or SEQ ID NO: 156 of 90% or more, particularly 95% or more, especially 98% or more,
  • DNA encoding a polypeptide having 3-dehydroquinate synthase activity, 3-dehydroquinate dehydratase activity, shikimate dehydrogenase activity, shikimate kinase activity, EPSP synthase activity, or chorismate synthase activity, respectively, can also be used.
  • DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to SEQ ID NO: 153, 5, 6, 154, 155, or SEQ ID NO: 156, and each of 3- DNA encoding a polypeptide having dehydroquinate synthase activity, 3-dehydroquinate dehydratase activity, shikimate dehydrogenase activity, shikimate kinase activity, EPSP synthase activity, or chorismate synthase activity can also be used.
  • 3-dehydroquinate synthase activity is determined by known methods (Meudi, S. et al., Dehydroquinate synthase from Escherichia coli, and its substrate 3-deoxy-D-arabino-heptulosonic acid 7-phosphate. Methods. Enzymol. 142: 306 -314 (1987)).
  • a reaction comprising a crude enzyme solution of 50 mM potassium phosphate buffer (pH 7.0), 0.2 mM DAHP, 0.2 mM NAD + , 1 mM Cobalt (II) chloride ⁇ 6H 2 O, 3-dehydroquinate dehydratase
  • the reaction was initiated by adding the test enzyme solution to the mixture, and the absorbance at 234 nm due to 3-dehydroshikimate produced by the coupling reaction of 3-dehydroquinate synthase activity and 3-dehydroquinate dehydratase activity.
  • the activity produced by 1 ⁇ mol of 3-dehydroshikimic acid per minute at 33 ° C. is defined as 1 unit of DHQ synthase activity.
  • the enhancement of 3-dehydroquinate synthase activity of the transformant is confirmed by an increase in 3-dehydroquinate synthase activity in the cell extract of the transformant.
  • 3-Dehydroquinate dehydratase activity is performed according to a known method (Chaudhuri, S. et al., 3-Dehydroquinate dehydratase from Escherichia coli. Methods. Enzymol. 142: 320-324 (1987)). That is, at 33 ° C., the reaction is started by adding the test enzyme solution to a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 7.0) and 0.5 mM 3-dehydroquinic acid, and 3-dehydroshiki-mi produced.
  • 1 unit of 3-dehydroquinic acid dehydratase activity is defined as 1 unit of 3-dehydroquinic acid dehydratase activity at 33 ° C per minute, and it is determined that 3-dehydroquinic acid dehydratase activity exists when activity is detected .
  • the enhancement of 3-dehydroquinate dehydratase activity of the transformant is confirmed by an increase in 3-dehydroquinate dehydratase activity in the cell extract of the transformant.
  • Shikimate dehydrogenase activity is measured according to a known method (Chaudhuri, S. et al., Shikimate dehydrogenase from Escherichia coli. Methods. Enzymol. 142: 315-320 (1987)).
  • the reaction was started by adding the test enzyme solution to a reaction mixture consisting of 100 mM Tris-HCl buffer (pH 7.5), 0.2 mM NADPH, 0.5 mM 3-dehydroshikimic acid, and NADPH
  • the activity at which 1 ⁇ mol of NADPH is consumed per minute at 33 ° C. is defined as 1 unit of shikimate dehydrogenase activity.
  • Shikimate kinase activity is measured according to a known method (Cheng, WC. Et al., Structures of Helicobacter pylori shikimate kinase reveal a selective inhibitor-induced-fit mechanism. PLos One. 7: e33481 (2012)).
  • the chorismate synthase activity is measured according to a known method (Kitzing, K. et al., Spectroscopic and Kinetic Characterization of the Bifunctional Chorismate Synthase from Neurospora crassa. J. Biol. Chem. 276: 42658-42666 (2001)).
  • the reduction of FMN can be performed by adding 5 mM dithionite or 1 mM NADPH.
  • the activity produced by 1 ⁇ mol anthranilic acid per minute at 37 ° C. is defined as 1 unit of the chorismate synthase activity.
  • the enhancement of the chorismate synthase activity of the transformant is confirmed by an increase in the chorismate synthase activity in the cell extract of the transformant.
  • Protocatechuic acid 3,4-dioxygenase is an enzyme that catalyzes the conversion of protocatechuic acid to ⁇ -carboxy-cis, cis muconic acid by ring opening of protocatechuic acid in the catabolic metabolic pathway of protocatechuic acid.
  • Protocatechuate 3,4-dioxygenase activity can be eliminated, inhibited, or decreased by disruption, deletion, or mutation of the protocatechuate 3,4-dioxygenase gene on the chromosome.
  • An example of the protocatechuate 3,4-dioxygenase gene of Corynebacterium glutamicum is pcaHG.
  • the fact that the protocatechuate 3,4-dioxygenase activity of the transformant is lost, inhibited, or reduced indicates that the protocatechuate 3,4-dioxygenase activity in the cell extract of the transformant is reduced. Measured and confirmed by the decrease or disappearance of the enzyme activity.
  • the activity at which 1 ⁇ mol of protocatechuic acid disappears in 1 minute at 33 ° C. is defined as 1 unit of protocatechuic acid 3,4-dioxygenase activity, and when the enzyme activity is detected, protocatechuic acid 3,4-dioxygenase activity Judge that there is.
  • sugar Phosphotransferase system is the uptake of sugars such as glucose into the cell and the phosphorus of sugars. It is a sugar transport mechanism existing only in prokaryotes, characterized by performing oxidation in a coupled manner. In Escherichia coli and coryneform bacteria, PTS plays a major role in the uptake of sugar into cells. PTS is a common protein, Enzyme I (PEP Protein kinase), HPr (Histidine-phosphorylatable protein), and a membrane protein involved in the specific transport of various sugars.
  • Enzyme I PEP Protein kinase
  • HPr Histidine-phosphorylatable protein
  • Phosphoenolpyruvic acid composed of glycoprotein II (enzyme II), is used as a phosphate donor and transports sugar into the cell as a phosphorylated form via a phosphate relay between these components System.
  • PTS consumes PEP, which is one of the common precursors of aromatic compounds, as a phosphate donating group for producing glucose-6-phosphate as glucose is transported into cells.
  • PEP is a key precursor compound in the production of aromatic compounds, and for the high production of aromatic compounds containing protocatechuic acid, the consumption of PEP by competitive metabolic pathways such as PTS is suppressed, and the production route to aromatic compounds is reduced. It is important to increase the availability of PEP.
  • the sugar uptake through PTS is inactivated, and at the same time, the PEP is not consumed with the sugar transport, via a sugar transport system (non-PTS sugar transport system) different from PTS. It is preferable that the sugar availability is provided.
  • the uptake of sugar into cells via PTS can be eliminated, inhibited or reduced by disruption, deletion or mutation of the gene encoding PTS on the chromosome of coryneform bacteria.
  • the gene encoding PTS include ptsI encoding Enzyme I, ptsH encoding Hpr, and ptsG encoding Enzyme II.
  • the ptsH gene encoding the Hpr protein, a common component of PTS is disrupted, It is preferably deleted or mutated.
  • a deletion type gene is generated by deleting a partial sequence of the gene and modified so as not to produce a normally functioning protein, and a bacterium is transformed with the DNA containing the gene so that the deletion type gene and the chromosome are on the chromosome.
  • a bacterium is transformed with the DNA containing the gene so that the deletion type gene and the chromosome are on the chromosome.
  • the gene on the chromosome can be replaced with a deletion or destruction type gene. Even if a protein encoded by a deletion-type or disruption-type gene is produced, it has a three-dimensional structure different from that of a wild-type protein, and its function is reduced or lost.
  • the ability of the coryneform bacterium transformant to lose, inhibit, or reduce the sugar transport ability via PTS means that sugars (glucose, sucrose, fructose, etc.) transported by PTS in the transformant. ) Is eliminated, inhibited or suppressed, and such phenotype is restored by introduction of a normal pts gene.
  • Corynebacterium glutamicum is a sugar transporter different from PTS, and there is a non-PTS glucose transporter that does not consume PEP due to intracellular transport of sugar. To do. Corynebacterium glutamicum whose sugar uptake through PTS is inhibited by disruption of the pts gene, etc.
  • glucose uptake into cells and growth of bacteria using glucose as a carbon source are non-PTS glucose transporter activity and glucokinase It is desirable to improve by enhancing the activity. It is considered that this makes it possible to avoid the consumption of PEP accompanying glucose transport and to supply more PEP for biosynthesis of aromatic compounds such as shikimic acid.
  • the uptake of glucose into cells by a non-PTS glucose transporter can be achieved by introducing a gene encoding a non-PTS glucose transporter or by a mutation in a non-PTS glucose transporter gene (control sequence or coding region) on the chromosome of a coryneform bacterium.
  • the gene expression level can be enhanced by introduction or nucleotide sequence substitution, or the activity of the gene product can be enhanced. Of these, it is simple and efficient to enhance the glucose uptake activity by introducing a non-PTS glucose transporter gene.
  • the origin of the non-PTS glucose transporter gene to be introduced is not particularly limited, but it is preferably a gene of Corynebacterium bacteria, particularly Corynebacterium glutamicum, in terms of good shikimic acid production efficiency.
  • Any non-PTS glucose transporter may be used as long as it functions in coryneform bacteria, such as inositol transporter (IolT1, IolT2) derived from Corynebacterium glutamicum, galactose permease (GalP) derived from E. coli (Escherichia coli), and zyomonas.
  • inositol transporter IolT1, IolT2
  • GalP galactose permease
  • E. coli Erscherichia coli
  • zyomonas examples include glucose facilitator (Glf) derived from Zymomonas mobilis.
  • glucose facilitator Glf
  • an example of the inositol transporter gene derived from Corynebacterium glutamicum is DNA (iolT1) having the base sequence of SEQ ID NO: 157.
  • a DNA comprising a nucleotide sequence having 90% or more, 95% or more, particularly 98% or more identity with SEQ ID NO: 157, and a DNA encoding a polypeptide having inositol transporter activity is also included.
  • a DNA that hybridizes with a DNA comprising a base sequence complementary to SEQ ID NO: 159 under stringent conditions and encodes a polypeptide having inositol transporter activity can also be used.
  • the protein encoded by DNA is a non-PTS glucose transporter, which means that a host cell that has lost its PTS-dependent glucose transport ability due to disruption of the ptsH gene, etc., and has reduced growth using glucose as a carbon source.
  • the growth of the transformant into which the DNA has been introduced and expressed, using glucose as a carbon source, or the rate of glucose consumption being higher than that of the cell before transformation, and the effect of the pts gene disruption It is confirmed as an index that it is not affected by the inhibition of PTS-dependent sugar transport.
  • the non-PTS glucose transporter activity of the transformant is enhanced because the growth using glucose as a carbon source or the glucose consumption rate in the transformant deficient in sugar transport by PTS. In the transformant, it is confirmed as an index that it is higher than that before gene introduction.
  • Glucokinase is an enzyme that catalyzes the conversion of glucose to glucose-6-phosphate.
  • the glucokinase activity is enhanced simultaneously with the enhancement of non-PTS glucose transporter-dependent glucose transport. This is characterized in that glucose uptake into cells and the subsequent glycolysis in the glycolysis and pentose / phosphate pathways are promoted.
  • the glucokinase activity is increased by high expression by introduction of the glucokinase gene, or by introduction of mutations into the glucokinase gene (regulatory sequence and gene coding region) on the chromosome, or by substitution of the sequence, It can be enhanced by increasing the activity of the product.
  • glucokinase genes cgR_2067 (glk1), cgR_2552 (glk2), and cgR_1739 (ppgK), on the chromosome of Corynebacterium glutamicum R strain.
  • cgR_2067 (glk1) and cgR_2552 (glk2) have high homology with glucokinase which uses ATP as a good substrate
  • cgR_1739 (ppgK) has high homology with glucokinase which uses polyphosphate as a good substrate.
  • one or more of these glucokinase genes are preferably enhanced, and more preferably all three are enhanced.
  • the enhancement of glucokinase activity is simple and efficient by introducing a glucokinase gene.
  • the origin of the glucokinase gene to be introduced is not particularly limited, it is preferably a gene of Corynebacterium, particularly Corynebacterium glutamicum, in terms of good shikimic acid production efficiency.
  • Examples of the glucokinase gene derived from Corynebacterium glutamicum include DNAs having the nucleotide sequences of SEQ ID NOs: 158, 159, and 160 (glk1, glk2, and ppgK, respectively).
  • a polypeptide comprising a nucleotide sequence having the identity of SEQ ID NO: 158, 159 or 160 with 90% or more, particularly 95% or more, especially 98% or more, and having a glucokinase activity DNA encoding can also be used.
  • a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to SEQ ID NO: 158, 159, or 160 and encodes a polypeptide having glucokinase activity. it can.
  • Glucokinase activity is a reaction mixture consisting of 100 mM Tris-HCl buffer (pH 7.5), 4 mM magnesium chloride, 1 mM ATP, 0.2 mM NADP + , 20 mM glucose, 1 U glucose-6-phosphate dehydrogenase at 33 ° C.
  • the activity at which 1 ⁇ mol of NADPH is produced per minute at 33 ° C. is defined as 1 unit of glucokinase activity.
  • GAPDH activity-enhanced glyceraldehyde-3-phosphate dehydrogenase is an enzyme that converts glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate.
  • GAPDH activity is preferably enhanced.
  • a coryneform bacterium transformant in which the pts gene is disrupted and the sugar uptake activity via non-PTS glucose transporter and the glucokinase activity are enhanced is a glycolytic metabolic intermediate during culture and reaction.
  • Dihydroxyacetone (DHA) which is a metabolite obtained by dephosphorylating dihydroxyacetone phosphate, and glycerol produced by further metabolism of DHA are remarkably accumulated.
  • intracellular concentrations of glyceraldehyde-3-phosphate and an upstream glycolytic metabolic intermediate are significantly increased in the transformant.
  • reaction step catalyzed by GAPDH is the rate-determining rate of sugar metabolism in the glycolytic system in the transformant, and the high expression of GAPDH in the transformant results in sugar consumption.
  • the present inventor has found that the production of the target product is also promoted. Therefore, in the present invention, it is desirable that the GAPDH activity of the transformant is enhanced to release the rate-limiting factor of sugar metabolism to promote sugar consumption and improve protocatechuic acid production ability.
  • GAPDH activity is high expression by introduction of the GAPDH gene, increase of gene expression level by introduction of mutation in GAPDH gene (control sequence and gene coding region) on the chromosome, or sequence substitution, or activity of the gene product It can be strengthened by increasing. Among them, the enhancement of GAPDH activity is simple and efficient when carried out by introducing the GAPDH gene.
  • the origin of the GAPDH gene to be introduced is not particularly limited, but it is preferably a gene of Corynebacterium, particularly Corynebacterium glutamicum, in terms of good protocatechuic acid production efficiency.
  • Examples of the GAPDH gene derived from Corynebacterium glutamicum include DNA (gapA) consisting of the base sequence of SEQ ID NO: 161.
  • a DNA comprising a nucleotide sequence having 90% or more, more than 95%, more preferably 98% or more identity with SEQ ID NO: 161, and a DNA encoding a polypeptide having GAPDH activity is also used.
  • a DNA that hybridizes with a DNA having a base sequence complementary to SEQ ID NO: 161 under stringent conditions and encodes a polypeptide having GAPDH activity can also be used.
  • the fact that the protein encoded by DNA is GAPDH is confirmed by measuring the GAPDH activity of the polypeptide encoded by the DNA.
  • GAPDH activity was measured at 33 ° C. using 25 mM phosphate buffer (pH 7.5), 25 mM trisethanolamine (pH 7.5), 0.2 mM EDTA, 5 mM NAD + , 5 mM glyceraldehyde-3-phosphate, The reaction is started by adding an enzyme solution to the reaction mixture consisting of By doing. The activity that 1 ⁇ mol of NADH is produced per minute at 33 ° C. is defined as 1 unit of GAPDH activity.
  • the GAPDH activity of the coryneform bacterium transformant is enhanced by measuring the GAPDH activity in the cell extract of the coryneform bacterium transformant.
  • DHAP dephosphorylation enzyme catalyzes the conversion of DHAP to dihydroxyacetone (DHA) by dephosphorylation.
  • DHA dihydroxyacetone
  • the DHAP phosphatase activity is preferably lost, inhibited or decreased.
  • coryneform bacteria that take up and use sugar in cells depending on the highly expressed non-PTS glucose transporter and glucokinase produce high amounts of DHA as a by-product. For this reason, it becomes possible to supply more carbon for production of aromatic compounds such as protocatechuic acid by blocking the DHA production pathway.
  • Corynebacterium glutamicum has HAD (haloacid dehalogenase) superfamily phosphatase (HdpA) as an enzyme that catalyzes the dephosphorylation of DHAP (Jojima, T. et. Al., Identification of a HAD superfamily phosphatase, HdpA , involved in 1,3-dihydroxyacetone production during sugar catabolism in Corynebacterium glutamicum. FEBS. Lett. 586: 4228-4232 (2012)).
  • the DHAP phosphatase activity of Corynebacterium glutamicum can be eliminated, inhibited or reduced by disruption, deletion or mutation of the DHAP phosphatase gene (hdpA) on the chromosome.
  • the fact that the DHAP phosphatase activity of the transformant is lost, inhibited, or decreased is determined by measuring the DHAP phosphatase activity in the cell extract of the transformant.
  • Inorganic phosphate ions liberated from DHAP according to known methods (Gawronski, JD, et. Al., Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection. Anal. Biochem. 327: 114-118 (2004)) Measure by colorimetric determination. When this quantitative value decreases or disappears, it is determined that the dihydroxyacetone phosphate phosphatase activity has disappeared, inhibited, or decreased.
  • each protein or DNA encoding the enzyme is integrated into the host chromosome, or They can be cloned into an appropriate vector that can be amplified in the host and introduced into the host.
  • the plasmid vector may be any plasmid vector as long as it contains a gene that controls the autonomous replication function in coryneform bacteria. Specific examples thereof include pAM330 derived from Brevibacterium lactofermentum 2256 (Japanese Patent Laid-Open No. 58-67699), [Miwa, K. et al., Cryptic plasmids in glutamic acid-producing bacteria. Agric Biol.
  • Preferred promoters include a promoter PgapA of glyceraldehyde 3-phosphate dehydrogenase A gene (gapA) derived from Corynebacterium glutamicum R, a promoter Pmdh of malate dehydrogenase gene (mdh), and a promoter PldhA of lactate dehydrogenase A gene (ldhA) Among them, PgapA is preferable.
  • Preferred terminators include the rrnB T1T2 terminator of the E. coli rRNA operon, the trpA terminator of E. coli, the trp terminator of Brevibacterium lactofermentum, and the rrnB T1T2 terminator is preferred.
  • Transformation transformation methods can be used without limitation any known methods.
  • known methods include calcium chloride / rubidium chloride method, calcium phosphate method, DEAE-dextran mediated transfection, and electric pulse method.
  • the electric pulse method is suitable for coryneform bacteria, and the electric pulse method can be performed by a known method (Kurusu, Y. et al., Electroporation-transformation system for Coryneform bacteria by auxotrophic complementation. Agric Biol. Chem. 54: 443-447 (1990)).
  • a natural medium or a synthetic medium usually containing a carbon source, a nitrogen source, inorganic salts, and other nutrient substances can be used.
  • carbon sources examples include glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol, glycerin and other sugars or sugar alcohols; acetic acid, citric acid, lactic acid, fumaric acid, maleic acid Or organic acids, such as gluconic acid; Alcohol, such as ethanol and a propanol, is mentioned.
  • a carbon source can be used individually by 1 type, or may mix 2 or more types. The concentration of these carbon sources in the medium is usually about 0.1 to 10 (w / v%).
  • the nitrogen source examples include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, urea, aqueous ammonia, sodium nitrate, and potassium nitrate. Further, corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolyzate, nitrogen-containing organic compounds such as amino acids, and the like can be used. As the nitrogen source, one kind may be used alone, or two or more kinds may be mixed and used. The nitrogen source concentration in the medium varies depending on the nitrogen compound used, but is usually about 0.1 to 10 (w / v%).
  • inorganic salts examples include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. These inorganic salts may be used alone or in a combination of two or more. The concentration of inorganic salts in the medium varies depending on the inorganic salt used, but is usually about 0.01 to 1 (w / v%).
  • the nutrient substance examples include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, defatted soy hydrochloride hydrolyzate, or extracts of animal or plant or microbial cells and their degradation products. However, it is usually about 0.1 to 10 (w / v%).
  • vitamins can be added as necessary. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like.
  • the pH of the medium is preferably about 6-8.
  • a preferred microorganism culture medium is A medium (Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions.J. Mol. Microbiol. Biotechnol. 7: 182-196 (2004)).
  • BT medium [Omumasaba, CA et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8: 91-103 (2004)].
  • the culture temperature may be about 15 to 45 ° C.
  • the culture time may be about 1 to 7 days.
  • Protocatechu is produced by a method comprising a step of producing protocatechuic acid or a salt thereof by culturing or reacting the transformant of the present invention described above in a reaction solution containing a saccharide.
  • An acid or a salt thereof can be produced.
  • Glucose is preferred as the saccharide, but in addition to monosaccharides such as fructose, mannose, arabinose, xylose and galactose, saccharides capable of producing glucose by metabolism can also be used.
  • Such sugars include oligosaccharides or polysaccharides having glucose units, disaccharides such as cellobiose, sucrose (sucrose), lactose, maltose, trehalose, cellobiose, xylobiose; polysaccharides such as dextrin or soluble starch, etc. Is mentioned.
  • molasses can also be used as a raw material containing these raw material compounds.
  • non-edible agricultural waste such as straw (rice straw, barley straw, wheat straw, rye straw, oat straw), bagasse, corn stover, energy crops such as switchgrass, napiergrass and miscanthus, and wood chips
  • a saccharified solution containing a plurality of sugars such as glucose obtained by saccharifying used paper with a saccharifying enzyme or the like can also be used.
  • the transformant Prior to the culture in a medium containing microbial growth saccharides, that is, the reaction, the transformant is preferably grown under aerobic conditions at a temperature of about 25 to 38 ° C. for about 12 to 48 hours.
  • a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, and other nutrient substances can be used.
  • carbon sources sugars (monosaccharides such as glucose, fructose, mannose, xylose, arabinose, galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, trehalose; polysaccharides such as starch; molasses etc.), Sugar alcohols such as mannitol, sorbitol, xylitol, glycerin; organic acids such as acetic acid, citrate fermentation, lactic acid, fumaric acid, maleic acid and gluconic acid; alcohols such as ethanol and propanol; hydrocarbons such as normal paraffin Can also be used.
  • a carbon source can be used individually by 1 type or in mixture of 2 or
  • inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate, urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like can be used.
  • corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolyzate, nitrogen-containing organic compounds such as amino acids, and the like can also be used.
  • a nitrogen source can be used individually by 1 type or in mixture of 2 or more types. The concentration of the nitrogen source in the medium varies depending on the nitrogen compound to be used, but is usually about 0.1 to 10 (w / v%).
  • inorganic salts examples include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate.
  • One inorganic salt can be used alone, or two or more inorganic salts can be mixed and used.
  • the concentration of inorganic salts in the medium varies depending on the inorganic salt used, but is usually about 0.01 to 1 (w / v%).
  • Examples of nutritional substances include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, defatted soy hydrochloride hydrolyzate, extracts of animals and plants or microbial cells, and degradation products thereof.
  • concentration of the nutrient substance in the medium varies depending on the nutrient substance used, but is usually about 0.1 to 10 (w / v%).
  • vitamins can be added as necessary. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like.
  • the pH of the medium is preferably about 6-8.
  • a medium (Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7: 182- 196 (2004)), BT medium (Omumasaba, CA et al., YneCorynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8: 91-103 (2004)) Etc.
  • the saccharide concentration may be used within the above range.
  • a culture solution or a reaction solution a natural reaction solution or a synthetic reaction solution containing a carbon source, a nitrogen source, inorganic salts, and the like can be used.
  • the carbon source the saccharide described above or molasses or saccharified solution containing the saccharide may be used.
  • a carbon source in addition to sugars, sugar alcohols such as mannitol, sorbitol, xylitol, glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, gluconic acid; ethanol, propanol, etc.
  • Non-alcoholic; hydrocarbons such as normal paraffin can also be used.
  • a carbon source can be used individually by 1 type or in mixture of 2 or more types.
  • the concentration of the saccharide as the raw material compound in the reaction solution is preferably about 1 to 20 (w / v%), more preferably about 2 to 10 (w / v%), and about 2 to 5 (w / v%). Is even more preferred. Further, the concentration of the total carbon source including the saccharide as a raw material may be about 2 to 5 (w / v%).
  • inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate, urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like can be used.
  • corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolyzate, nitrogen-containing organic compounds such as amino acids, and the like can also be used.
  • a nitrogen source can be used individually by 1 type or in mixture of 2 or more types. The concentration of the nitrogen source in the reaction solution varies depending on the nitrogen compound to be used, but is usually about 0.1 to 10 (w / v%).
  • inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate.
  • One inorganic salt can be used alone, or two or more inorganic salts can be mixed and used.
  • the concentration of the inorganic salt in the reaction solution varies depending on the inorganic salt used, but is usually about 0.01 to 1 (w / v%).
  • vitamins can be added as necessary.
  • vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like.
  • the pH of the reaction solution is preferably about 6-8.
  • reaction solution for coryneform bacteria include the BT medium described above.
  • saccharide concentration may be used within the above range.
  • the culture temperature or reaction temperature that is, the survival temperature of the transformant is preferably about 20 to 50 ° C., more preferably about 25 to 47 ° C. If it is the said temperature range, protocatechuic acid can be manufactured efficiently.
  • the culture or reaction time is preferably about 1 to 7 days, more preferably about 1 to 3 days.
  • the culture may be any of batch type, fed-batch type, and continuous type. Among these, the batch type is preferable.
  • the reaction may be carried out under aerobic conditions or under reducing conditions. The ability of the transformant of the present invention to produce protocatechuic acid or a salt thereof is higher under aerobic conditions.
  • Not proliferating in the present invention includes substantially not proliferating or hardly proliferating.
  • a reaction solution lacking or limiting one or more of vitamins such as biotin and thiamine, which are essential compounds for the growth of microorganisms, nitrogen sources, or amino acids essential for the growth of auxotrophic transformants By using, the growth of the transformant can be avoided or suppressed.
  • the reduction condition is defined by the oxidation-reduction potential of the reaction solution.
  • the oxidation-reduction potential of the reaction solution is preferably about ⁇ 200 mV to ⁇ 500 mV, more preferably about ⁇ 150 mV to ⁇ 500 mV.
  • the reduction state of the reaction solution can be estimated simply with a resazurin indicator (decolorization from blue to colorless in the reduction state), but using a redox potentiometer (for example, BROADLEY JAMES, ORP Electrodes) Can be measured.
  • a known method can be used without limitation.
  • an aqueous solution for reaction solution may be used as a liquid medium for the reaction solution instead of distilled water, etc.
  • the method for adjusting the aqueous solution for reaction solution is, for example, a culture solution preparation for absolute anaerobic microorganisms such as sulfate-reducing microorganisms.
  • an aqueous solution for reaction solution under reducing conditions can be obtained by removing dissolved gas by heat treatment or decompression treatment of distilled water or the like.
  • distilled water or the like is treated for about 1 to 60 minutes, preferably about 5 to 40 minutes under reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less.
  • the dissolved gas, particularly dissolved oxygen can be removed to prepare an aqueous solution for reaction solution under reducing conditions.
  • an appropriate reducing agent for example, thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, etc.
  • An appropriate combination of these methods is also an effective method for preparing an aqueous solution for reaction solution under reducing conditions.
  • the reaction solution is preferably maintained under reducing conditions during the reaction.
  • the reaction system is made of an inert gas such as nitrogen gas or carbon dioxide gas.
  • the method of enclosing is mentioned.
  • a pH maintenance adjusting solution of the reaction system or various nutrient solution in such a case, it is effective to remove oxygen from the added solution in advance.
  • protocatechuic acid or a salt thereof is produced in the culture solution or reaction solution.
  • the salt of protocatechuic acid varies depending on the medium or components of the reaction solution, and examples thereof include alkali metal salts (sodium salt, potassium salt, etc.) and alkaline earth metal salts (magnesium salt, calcium salt, etc.).
  • chromosomal DNA was prepared from the following strains. Corynebacterium glutamicum R (FERM P-18976), Escherichia coli K-12 MG1655, Providencia rustigianii JCM 3953, Corynebacterium casei JCM 12072, Corynebacterium casei JCM 12072 Efficiens (Corynebacterium efficiens NBRC 100395), Pantoea ananatis (Pantoea ananatis LMG 20103), Gluconobacter oxydans ATCC 621H, Pseudomonas putida NBRC 14164, Rhodosdom R , Acinetobacter baylyi ATCC33305, Alteromonas macleodii NBRC 102226, Marinobacter hydrocarbonoclasticus JCM 20777), Methynebacterium glutamicum R (FERM P-18976), Escherichia coli K-12 MG1655, Providencia rustigianii
  • Table 2 shows the introduced cloning vectors and the obtained plasmid names. Since tkt and tal (tkt-tal gene; SEQ ID NO: 1), aroC and aroK and aroB (aroCKB; SEQ ID NO: 3) are arranged in the same direction on the chromosome, cloning was performed together. .
  • pCRB260, pCRB263, pCRB266, pCRB267, pCRB274 and pCRB279 introduced a restriction enzyme site (unique site) for incorporating a gene into the SSI region by inverse PCR.
  • Table 3 shows primer sequences used for isolation and inverse PCR of the SSI region and the resulting vector for chromosome introduction.
  • the PgapA promoter fusion enzyme gene fragment was obtained from the PCA production-related gene expression plasmid constructed in Table 2 and introduced into the above-described plasmid for chromosome introduction.
  • the obtained plasmids for PCA production-related gene chromosome introduction are shown in Table 4.
  • the markerless chromosomal gene transfer vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R.
  • the double crossover strain shows kanamycin sensitivity due to loss of the kanamycin resistance gene on pCRA725 and growth in a sucrose-containing medium due to loss of the sacR-sacB gene.
  • the markerless chromosome gene-introduced strain exhibits kanamycin sensitivity and sucrose-containing medium growth.
  • a PCA production-related gene chromosome introduction strain was constructed using the above-mentioned plasmid for PCA production-related gene chromosome introduction and the plasmid for chromosome gene disruption.
  • a host strain a xylose cellobiose assimilating coryneform bacterium Corynebacterium glutamicum X5C1 strain [Appl Microbiol Biotechnol. 81 (4): 691-699 (2008)] was used.
  • plasmid pCRA728 J Mol Microbiol Biotechnol.
  • Corynebacterium glutamicum PCA4 was deposited internationally at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamashi, Kisarazu, Chiba, Japan (zip code 292-0818) ( Date of international deposit under the Budapest Treaty: March 9, 2016, deposit number: NITE BP-02217). This stock is publicly available.
  • Corynebacterium glutamicum which is preferable as a host microorganism in the present invention
  • to protocatechuic acid in comparison with other microorganisms, Corynebacterium glutamicum, Escherichia coli, Bacillus subtilis, Pseudomonas putida, Rhodococcus erythropolis, and Saccharomyces cerevisiae The growth inhibitory effect of protocatechuic acid in aerobic culture was investigated.
  • Corynebacterium glutamicum R strain grown on the above plate was mixed with A liquid medium containing 4% glucose [(NH 2 ) 2 CO 2 g, (NH 4 ) 2 SO 4 7 g, KH 2 PO 4 0.5 g, K 2 HPO 4 0.5 g, MgSO 4 ⁇ 7H 2 O 0.5 g, 0.06% (w / v) Fe 2 SO 4 ⁇ 7H 2 O + 0.042% (w / v) MnSO 4 ⁇ 2H 2 O 1 ml, 0.02% (w / v) biotin solution 1 ml, 0.01% (w / v) thiamine solution 2 ml, yeast extract 2 g, vitamin assay casamino acid 7 g dissolved in 1 L of distilled water] After inoculation, the cells were cultured under aerobic shaking at 33 ° C for 16 hours.
  • Escherichia coli K12, Bacillus subtilis NBRC14144, Pseudomonas putida ATCC700801, and Rhodococcus erythropolis ATCC27854 to LB agar medium [1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar], respectively.
  • the Escherichia coli K12 strain and the Bacillus subtilis NBRC14144 strain were cultured at 37 ° C, and the Pseudomonas putida ATCC700801 strain and the Rhodococcus erythropolis ATCC27854 strain were cultured at 30 ° C for 16 hours.
  • Each strain grown on the above plate was inoculated into 10 ml of LB liquid medium [1% polypeptone, 0.5% yeast extract, and 0.5% sodium chloride], Escherichia coli K12 strain, and Bacillus subtilis NBRC14144 strain at 37 ° C, Pseudomonas putida ATCC700801 strain and Rhodococcus erythropolis ATCC27854 strain were aerobically cultured at 30 ° C. for 16 hours.
  • Escherichia coli K12 strain and Bacillus subtilis NBRC14144 strain were cultured at 37 ° C, and Pseudomonas putida ATCC700801 strain and Rhodococcus erythropolis ATCC27854 strain were aerobically cultured at 30 ° C. Proliferation of cells was carried out by measuring the OD 610.
  • Saccharomyces cerevisiae NBRC2376 strain was applied to YPD agar medium [2% polypeptone, 1% yeast extract, 2% glucose, and 1.5% agar] and cultured at 30 ° C. for 16 hours. Saccharomyces cerevisiae NBRC2376 strain grown on the above plate was inoculated into YPD liquid medium [2% polypeptone, 1% yeast extract, and 2% glucose], and aerobically shaken and cultured at 30 ° C. for 16 hours. .
  • FIG. 2 shows the results of examining the influence of protocatechuic acid addition to the medium on the aerobic growth of each strain.
  • the Escherichia coli K12 strain was significantly inhibited in the presence of 100 mM protocatechuic acid, and the growth was almost completely inhibited at 250 mM.
  • Bacillus subtilis NBRC14144 strain was markedly inhibited in the presence of 250 mM protocatechuic acid, and growth was almost completely inhibited at 500 mM.
  • Pseudomonas putida ATCC700801 strain was strongly inhibited in the presence of 100 mM protocatechuic acid, and growth was almost completely inhibited at 250 mM.
  • Rhodococcus erythropolis ATCC27854 strain was strongly inhibited in the presence of 250 mM protocatechuic acid, and growth was almost completely inhibited at 500 mM.
  • Saccharomyces cerevisiae NBRC2376 strain was inhibited from growth in the presence of 250 mM protocatechuic acid and markedly inhibited at 500 mM.
  • the Corynebacterium glutamicum R strain was capable of vigorous growth even in the presence of 250-500 mM protocatechuic acid, in which the growth of other strains was markedly or almost completely inhibited.
  • Corynebacterium glutamicum has high resistance to protocatechuic acid compared to other microorganisms that have been reported as protocatechuic acid production hosts and representative solvent-resistant bacteria, and therefore has high suitability as a protocatechuic acid production host. It has been shown.
  • FIG. 3 shows the glucose consumption of Corynebacterium glutamicum R strain after 24 hours of culture in the presence of various concentrations of protocatechuic acid. As shown in FIG. 3, it can be seen that Corynebacterium glutamicum has a slight decrease in sugar consumption even in the presence of a high concentration of protocatechuic acid.
  • Corynebacterium is Corynebacterium glutamicum protocatechuic acid-producing strains of the mixed sugar utilization lines from protocatechuic acid production test Corynebacterium glutamicum R strain by aerobic stationary cell reaction under jar fermentor control of transformants were constructed as a base, PCA1 , PCA2, PCA3, PCA4, PCA5 strains (Example 1 (Table 6)), protocatechuic acid production ability in aerobic stationary cell reaction under the control of jar fermenter (Able Co., Ltd., model: BMJ1L) was confirmed according to the method described below.
  • the PCA1 strain was prepared by adding 10 ml of the A liquid medium (phenylalanine, tyrosine, tryptophan 20 ⁇ g / ml each, p-aminobenzoic acid 10 ⁇ g / ml, shikimic acid 3.2 mM, and glucose 4% (each final concentration) ( After inoculation in a test tube), and after inoculation in 10 ml of the A liquid medium (in a test tube) with 10% of glucose added to PCA2, PCA3, PCA4, and PCA5 strains at 33 ° C for 12-16 hours, Aerobic shaking culture was performed.
  • the A liquid medium phenylalanine, tyrosine, tryptophan 20 ⁇ g / ml each, p-aminobenzoic acid 10 ⁇ g / ml, shikimic acid 3.2 mM, and glucose 4% (each final concentration)
  • Corynebacterium glutamicum for PCA2, PCA3, PCA4, and PCA5 strains glucose 100 g / l, and antifoam (Dishome CB-442) 3 g / l (each final concentration) added, 600 ml
  • the A (-UB) liquid medium was inoculated so that OD 610 would be 0.3, and each was cultivated at 33 ° C., pH 7.0 (5 N ammonia water) with a 1000 ml capacity jar fermenter (manufactured by Able Corporation, model: BMJ1L).
  • the Corynebacterium glutamicum strain grown under the above conditions is collected by centrifugation (4 ° C, 5000 xg, 10 minutes), and the cells are collected in a BT (-UB) liquid medium [(NH 4 ) 2 SO 4 7 g, KH 2 PO 4 0.5 g, K 2 HPO 4 0.5 g, MgSO 4 ⁇ 7H 2 O 0.5 g, 0.06% (w / v) (Fe 2 SO 4 ⁇ 7H 2 O + 0.042% (w / v) MnSO 4 ⁇ 2H 2 O) 1 ml, 100 ⁇ g / ml thiamine solution 2 ml dissolved in 1 L of distilled water] washed once, then 25 g wet bacteria against the BT (-UB) liquid medium containing 10% glucose / 250 ml (10% of the microbial cells are present in the medium as wet cell weight) and suspended using the 1000 ml jar fermenter at 33 ° C, pH 7.0 (addition of
  • the glucose concentration in the reaction solution was measured over time using a glucose sensor (Oji Scientific Instruments, BF-5i), and glucose was added as necessary.
  • Aromatic metabolite concentrations in the cell reaction supernatant were determined by high-performance liquid chromatography (Prominence HPLC device (manufactured by Shimadzu Corporation), COSMOSIL Packed column 5C18-AR-II. Acid)].
  • Table 8 shows the results of the protocatechuic acid production experiment by the aerobic stationary cell reaction using each strain.
  • PCA production amount of each strain 24 hours after the reaction PCA1 strain 273 mM (42.1 g / l), PCA2 strain 153 mM (23.6 g / l), PCA3 strain 515 mM (79.4 g / l), PCA4 The strain was 536 m (82.5 g / l), and the PCA5 strain was 408 m (62.8 g / l).
  • the molar yield of sugars for PCA production in each strain was 33.8% for PCA1 strain, 10.0% for PCA2 strain, 34.6% for PCA3 strain, 39.3% for PCA4 strain, and 30.2% for PCA5 strain.
  • PCA3 strain into which Corynebacterium glutamicum 3-dehydroshikimate dehydratase gene was introduced and the PCA4 strain into which Corynebacterium halotolerance 3-dehydroshikimate dehydratase gene was introduced showed particularly high PCA productivity.
  • these PCA3, PCA4, and PCA5 strains were shown to proliferate vigorously without the addition of supplemental nutrient sources containing aromatic amino acids even when the cells were cultured in a nutrient medium.
  • PCA1 strains that depend only on protocatechuic acid production by conversion of 3-dehydroshikimate to protocatechuic acid catalyzed by 3-dehydroshikimate dehydratase also showed a relatively high protocatechuic acid producing ability
  • the productivity was inferior to that of PCA3, PCA4, or PCA5 strains that produce PCA from both pathways (a) and (b).
  • PCA3 strain shows the requirement for aromatic amino acids and 4-aminobenzoic acid, and this strain grows in nutrient medium. In the case of making them, it was necessary to add those nutrient sources to the medium.
  • Example 3 Measurement of enzyme activities of 3-dehydroshikimate dehydratase, chorismate pyruvate lyase, and 4-hydroxybenzoate hydroxylase in protocatechuic acid producing strains PCA1, PCA2, and PCA3
  • various enzyme activities of 3-dehydroshikimate dehydratase, chorismate pyruvate lyase, and 4-hydroxybenzoate hydroxylase were measured according to the following methods. Perform the aerobic static cell reaction using jar fermenters of CRZ22 strain, PCA1 strain, PCA2 strain, and PCA3 strain in the same procedure as in Example 1, and collect each strain culture solution 6 hours after the reaction.
  • the cells were collected by centrifugation. After washing the cells once with 20 mM Tris-HCl (pH 7.5), 1 ml of a cell disruption buffer (100 mM Tris-HCl (pH 7.5), 20 mM KCl, 20 mM MgCl 2 , 0.1 mM EDTA, and 2 mM DTT)) and crushed using a multi-bead shocker (Yasui Kikai) and glass beads.
  • the cell disruption solution was centrifuged at 15000 rpm, 10 min, 4 ° C. to obtain a supernatant fraction as a crude enzyme extract.
  • the protein concentration of each crude enzyme extract was quantified using Protein assay kit (Bio-Rad, USA) and BSA (Bovine serum albumin) as a standard. Each enzyme activity in the crude enzyme extract of each strain was measured according to the activity measurement method described above.
  • the parental CRZ22 strain did not detect significant activity in any of the three enzyme activities tested. This suggested that these enzymes were weakly expressed or hardly expressed in the strain.
  • 3-dehydroshikimate dehydratase (QsuB) activity was detected with the introduction of the 3-dehydroshikimate dehydratase gene (qsuB). Since the aroE gene encoding shikimate dehydrogenase is disrupted in the PCA1 strain, the shikimate pathway is blocked at the enzyme reaction stage. Therefore, it supports that PCA production in the PCA1 strain occurs dependently only on (a) 3-dehydroshikimate dehydratase.
  • Example 4 Search for a heterologous gene encoding a competent shikimate dehydratase From the results of Example 2 and Example 3, the productivity of protocatechuic acid by the coryneform bacterial transformant was determined by the enzyme activity of the 3-dehydroshikimate dehydratase gene introduced. It has been shown that the strength is significantly increased by strengthening. On the other hand, in the coryneform bacterial transformants PCA1 and PCA3, the 3-dehydroshikimate dehydratase gene derived from Corynebacterium glutamicum has been introduced, but the more effective 3-dehydroshikimate dehydratase has been introduced. The possibility of being present in microorganisms was also considered.
  • 3-dehydroshikimate dehydratase gene qsuB
  • shikimate dehydrogenase gene aroE
  • Cells grown under the above conditions were glucose 4%, phenylalanine, tyrosine, tryptophan 20 ⁇ g / ml each, p-aminobenzoic acid 10 ⁇ g / ml, shikimic acid 3.2 mM, and kanamycin 50 ⁇ g / ml (each final concentration) was inoculated into 10 ml of the above-mentioned A liquid medium (in a test tube) so that the OD 610 was 0.2, and the culture was shaken aerobically at 33 ° C. for 24 hours.
  • protocatechuic acid or a salt thereof can be produced from glucose or the like with practical efficiency using a microorganism.

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Abstract

La présente invention concerne un micro-organisme qui peut produire de manière efficace de l'acide protocatéchuique ou son sel en utilisant un sucre comme matière première, et un procédé de production de manière efficace d'acide protocatéchuique ou son sel utilisant le micro-organisme. Le micro-organisme est un transformant présentant une capacité de production d'acide protocatéchuique, qui a été soumis aux modifications (A), (B), et (C) : (A) amélioration de l'activité 3-déshydroshikimate déshydratase, (B) amélioration de l'activité chorismate pyruvate lyase, et (C) amélioration de l'activité 4-hydroxybenzoate hydroxylase. Le procédé de production d'acide protocatéchuique ou son sel comprend une étape de culture du transformant dans un liquide réactionnel contenant un sucre de sorte que le transformant produise de l'acide protocatéchuique ou son sel.
PCT/JP2017/007233 2016-03-28 2017-02-24 Transformant, et procédé de production d'acide protocatéchuique ou son sel l'utilisant WO2017169399A1 (fr)

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JP7217294B2 (ja) 2018-12-20 2023-02-02 公益財団法人地球環境産業技術研究機構 カルボニル化合物の製造法
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CN111471630A (zh) * 2020-06-11 2020-07-31 鲁东大学 一株棒杆菌Ytld-phe09及其应用
DE102021000394A1 (de) 2021-01-27 2022-07-28 Forschungszentrum Jülich GmbH Herstellung von 3,4-Dihydroxybenzoat aus D-Xylose mit coryneformen Bakterien
CN113981014A (zh) * 2021-08-30 2022-01-28 黄山科宏生物香料股份有限公司 生产原儿茶酸的方法

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CN109477066A (zh) 2019-03-15
US20190119664A1 (en) 2019-04-25
US10961526B2 (en) 2021-03-30
EP3438245A1 (fr) 2019-02-06
JP6685388B2 (ja) 2020-04-22

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